Abstract:

The invention is related to a method for stimulating carbon fixation in
plants by applying an aqueous solution of an oligo-carrageenan selected
from the group consisting of kappa1, kappa2, lambda or iota, obtained by
acid hydrolysis from commercial pure carrageenans extracted from seaweed.
Particularly, a method for stimulating carbon fixation in plant by
increasing the net photosynthesis and the activity of the enzyme 1,5
biphophate carboxylase/oxygenase ribluose (Rubisco) in plants. More
particularly, it is disclosed a method for stimulating carbon fixation in
tobacco, beet or other commercial variety. Even more particularly, a
method for stimulating carbon fixation on plants applying by spraying an
aqueous solution of such oligo-carrageenans on the plant or parts
thereof.

Claims:

1. Method for stimulating carbon fixation in plants comprising spraying
on plants or parts thereof an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa1, kappa2, lambda or iota.

2. The method of claim 1, wherein the oligo-carrageenan is selected from
the group consisting of kappa2, lambda or iota.

5. The method of claim 1 wherein the plants are selected from a
greenhouse or a field of growing plants.

6. The method of claim 2, wherein the plants are selected from a
greenhouse or a field growing tobacco plants.

7. The method of claim 3, wherein the plants are selected from a
greenhouse or a field of growing plants.

8. The method of claim 5, wherein the plant is selected from tobacco or
beet.

9. The method of claim 6, wherein the plant is selected from a greenhouse
growing tobacco or beet plant.

10. The method of claim 2, wherein the plant is selected from a field
growing tobacco plants.

11. The method of claim 3, wherein the plant is selected from a field
growing tobacco plants.

12. The method of claim 8, wherein the aqueous solution of
align-carrageenan is sprayed on the leaf of the tobacco or beet plant.

13. The method of claim 1, wherein the aqueous solution of
oligo-carrageenans has a concentration of 1 mg mL.sup.-1.

14. The method of claim 1, wherein the aqueous solution of
oligo-carrageenan is sprayed weekly or a total of three times during
growing and before flowering.

15. The method of claim 5, wherein the aqueous solution of
oligo-carrageenan is sprayed weekly or a total of three times during
growing and before flowering.

16. The method of claim 1, wherein the aqueous solution of
oligo-carrageenan is sprayed weekly or a total of four times before being
transferred to a field and growing up to flowering.

17. The method of claim 5, wherein the aqueous solution of
oligo-carrageenan is sprayed weekly or a total of four times before being
transferred to a field and growing up to flowering.

18. The method of claim 10, wherein the aqueous of oligo-carrageenan is
sprayed weekly or a total of four times before being transferred to a
field and growing up to flowering.

Description:

DESCRIPTIVE MEMORY

[0001] The present application is directed to a method that stimulate
carbon fixation in plants using an aqueous solution of oligo-carrageenans
selected from the group consisting of kappa1, kappa2, lambda or iota,
obtained by acid hydrolysis of pure commercial carrageenans extracted
from marine algae. In particular, a method to stimulate carbon fixation
in plants by enhancing net photosynthesis and activity of ribulose 1,5
biphosphate carboxilase/oxygenase (rubisco) in plants is described. In
addition, a method to stimulate carbon fixation in tobacco plants, or in
sugar beet plants, or in other plant varieties of commercial interest is
presented. Even more particularly, a method to stimulate carbon fixation
in plant by spraying a water solution of the mentioned oligo-carrageenans
on plants or their parts is mentioned. More particularly, a method to
stimulate carbon fixation in plants by spraying a water solution of
oligo-carrageenans obtained using the method described in the Chilean
patent application No 2369-2007 or its corresponding WO 022221-2009 is
described.

PREVIOUS ART

[0002] In the last decades, the exponential growth of human population,
the increase of urban and industrial areas, the decrease in cultivable
soils due to climate change and aridization and the increase of areas for
plant biofuel production (bioethanol and biodiesel) has lead to a
progressive decreased cultivable surfaces used for human and animal
feeding. Thus, products and/or methods to enhance plant productivity in
order to increase plant biomass and the number, size or quality of bulbs,
tubers, seeds and fruits are urgently required in order to ensure feeding
of future world population.

[0003] It is well known, that plant growth and development is mainly
determined by photosynthesis. This biological process allows fixation of
inorganic carbon (CO2) into organic molecules required for basal and
secondary metabolism in plants. In this process, the energy of light is
captured by the green pigment chlorophyll determining the lysis of a
water molecule which releases an electron that is transported to
photosystem II and photosystem I leading to the synthesis of reducing
power (NADPH).

[0004] Moreover, the lysis of a water molecule releases a proton
generating a gradient that activate ATP synthase which synthesize ATP.
Synthesized NADPH and ATP are used as substrates by Calvin cycle enzymes
such as ribulose 1,5 biphosphate carboxilase/oxygenase (rubisco) which
fixed a CO2 molecule and incorporate carbon to an organic molecule
of three carbons. Thus, the increase in net photosynthesis reflects an
increase in carbon fixation, the activation of rubisco and the
stimulation of basal and secondary metabolism in plants.

[0005] In this century, atmospheric CO2 has increased from 280
μmol/mol to 380 μmol/mol and will reach 550 μmol/mol in year
2050 (Solomon et al., 2007. Climate change, p. 996, Cambridge University
Press, UK). It is now accepted that the increase in atmospheric CO2
and its adverse effect on climate (greenhouse effect) resides on CO2
emissions of human industrial activities (antropogenic effect). Thus, the
enhancement of carbon fixation by the stimulation of photosynthesis in
plants will be highly beneficial to stabilize climate changes reducing
aridization of cultivable soils and to increase plant productivity.

[0006] Recently, the US Department of Agriculture (USDA) with several
research groups performed FACE (Free Air CO2-Enriched) field
experiments in different countries using a high concentration of CO2
(580 ppm) in order to stimulate carbon fixation in crops and trees
(Ainsworth et al, 2008 Next generation of elevated [CO2] experiments with
crops: a critical investment for feeding the future world. Plant cell and
Environment 31, 1317-1324). However, the yield of wheat, rice and soybean
plants increased only in 14% in average instead of 38% as was
theoretically predicted. In addition, experiments using classical
genetics performed during the past 20 years did not allow the isolation
of crop varieties with increased carbon fixation or growth and yield.

[0007] Until now, only few agents that stimulate carbon fixation in plants
have been reported such as a mixture of brassinosteroids (WO patent
application 125069-2008), a lipo-chito-oligosaccharide isolated from Gram
negative bacteria of the genus Rhizobium and Bradirhizobium (US patent
application 7550068-2007), a chito-oligosaccharide prepared from quitin
of arthropods (Chinese patent 1733657-2006), a mixture of short-chain
alcohols and aminoacids (US patent 00695-2003) and an alcoholic extract
from the plant Impatiens balsamina L. (Japanese patent 10109914-1998).

[0008] The present invention shows that oligo-carrageenans obtained by
acid depolymerization of pure commercial carrageenans extracted from
marine algae prepared following the method described in the Chilean
patent application No 2369-2007 and its corresponding WO 022221-2009
stimulate net photosynthesis and the activity of rubisco enzyme in
plants, in particular, tobacco plants var. Burley grown in a greenhouse
or in the field and sugar beets var. Fidelia grown in a greenhouse.

SHORT DESCRIPTION OF FIGURES

[0009]FIG. 1A: Effect of an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa1, kappa2, lambda and iota on
net photosynthesis in tobacco plants var. Burley grown in a greenhouse.

[0010] FIG. 1B: Effect of an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa1, kappa2, lambda and iota on
rubisco enzyme activity in tobacco plants var. Burley grown in a
greenhouse.

[0011]FIG. 2A: Effect of an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa1, kappa2, lambda and iota on
net photosynthesis in tobacco plants var. Burley grown in field.

[0012]FIG. 2B: Effect of an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa2, lambda and iota on rubisco
enzyme activity in tobacco plants var. Burley grown in field.

[0013]FIG. 3A: Effect of an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa2, lambda and iota on net
photosynthesis in sugar beet plants var. Fidelia grown in a greenhouse.

[0014]FIG. 3B: Effect of an aqueous solution of an oligo-carrageenan
selected from the group consisting of kappa1, kappa2, lambda and iota on
rubisco enzyme activity in sugar beet plants var. Fidelia grown in a
greenhouse.

[0016] The greenhouse experiment using tobacco plants was performed with
plants var. Burley (n=10) grown in garden bed and then in plastic bags
containing compost until they reached the height of 30 cm. Plant leaves
were sprayed with water (control) or with an aqueous solution of
oligo-carrageenan selected from the group consistent of kappa1, kappa2,
lambda and iota at a concentration of 1 mg mL-1, once a week, for
three weeks and then grown before flowering.

[0017] The field experiment using tobacco plants was performed with plants
var. Burley (n=30) grown in garden bed and then in plastic bags
containing compost until they reached the height of 15 cm. Plant leaves
were sprayed with water (control) or with an aqueous solution of
oligo-carrageenan selected from the group consistent of kappa1, kappa2,
lambda and iota at a concentration of 1 mg mL-1, once a week, for
four weeks and then grown before flowering.

[0018] The greenhouse experiment using sugar beet plants was performed
with plants var. Fidelia (n=10) grown in plastic bags containing compost
until they reached the height of 20 cm. Plant leaves were sprayed with
water (control) or with an aqueous solution of oligo-carrageenan selected
from the group consistent of kappa2, lambda and iota at a concentration
of 1 mg mL-1, once a week, for three weeks and then grown before
flowering.

[0019] Net photosynthesis was determined in tobacco plants grown in a
greenhouse (n=10), tobacco plants grown in field (n=30) and sugar beet
plants grown in a greenhouse (n=10) using an InfraRed Gas Analyzer (IRGA,
PPsystem, model Ciras-1) and detecting photosynthesis in 5 leaves of each
plant.

[0020] Rubisco activity was determined in tobacco plants grown in a
greenhouse (n=10), tobacco plants grown in field (n=10) and sugar beet
plants grown in a greenhouse (n=10) using a protein extract from plant
leaves and a coupled enzyme reaction containing phosphoglycerate-kinase
(an ATP consuming enzyme) and glyceraldehydes-3 phosphate dehydrogenase
(a NADPH consuming enzyme) and determining the decrease in absorbance at
340 nm due to NADPH consumption.

[0021] Results showed that net photosynthesis increased in tobacco and
sugar beet plants treated with oligo-carrageenans kappa2, lambda and
iota. In particular, the highest increase in net photosynthesis was
induced by oligo-carrageenan iota in tobacco plants grown in greenhouse,
or in the field, and in sugar beet plants and corresponds to 4.3, 4.9 and
1.7 times, respectively. On the other hand, rubisco activity increased in
tobacco and sugar beet plants treated with oligo-carragenans kappa1,
kappa2, lambda and iota. In particular, the highest increase in rubisco
activity was induced by oligo-carrageenan iota in tobacco plants grown in
greenhouse, or in the field, and in sugar beet plants and corresponds to
4.4, 4.5 and 5.4 times, respectively. These results indicate that carbon
fixation increased in tobacco and sugar beet plants treated with
oligo-carrageenans kappa1, kappa2, lambda and iota, mainly with
oligo-carrageenan iota. The increase in carbon fixation is due to the
increase in net photosynthesis and is reflected by the increase in
rubisco activity.

[0022] FIG. 1 shows the stimulation of net photosynthesis (FIG. 1A) and
the increase in rubisco activity (FIG. 1B) in tobacco plants and treated
with an aqueous solution of oligo-carrageenans selected from the group
consisting of kappa1, kappa2, lambda or iota and grown in greenhouse.
FIG. 2 shows the stimulation of net photosynthesis (FIG. 2A) and the
increase in rubisco activity (FIG. 2B) in tobacco plants treated with an
aqueous solution of oligo-carrageenans selected from the group consisting
of kappa1, kappa2, lambda or iota and grown in field. FIG. 3 shows the
stimulation of net photosynthesis (FIG. 3A) and the increase in rubisco
activity (FIG. 3B) in sugar beet plants and treated with an aqueous
solution of oligo-carrageenans selected from the group consisting of
kappa1, kappa2, lambda or iota and grown in greenhouse.

[0023] Tables Ito VI show data illustrated in FIGS. 1A, 1B, 2A, 2B, 3A and
3B, the increase in times of net photosynthesis and the increase in
specific activity of rubisco enzyme in tobacco and sugar beet plants
treated with oligo-carrageenans compared to the control.